# Solid Surface Vitrification Is Better than Slow Freezing for the Long-Term Preservation of Testicular Fragments from Prepubertal Collared Peccaries (Pecari tajacu Linnaeus, 1758)

## TL;DR

Solid surface vitrification with 6 M cryoprotectants is better than slow freezing for preserving testicular tissue from young collared peccaries.

## Contribution

The study identifies the optimal vitrification protocol for prepubertal collared peccary testicular fragments.

## Key findings

- SSV with 6 M cryoprotectants showed better histological integrity and lower cellular damage than SSV 3 M.
- SSV 6 M preserved DNA integrity as well as the control group and improved spermatogonia cell proliferation.
- Cell viability was lower in all treatments compared to the control group, but SSV 6 M was the most effective method.

## Abstract

Cryobanking is an essential tool in understanding and sustaining biodiversity. Among the multiple types of biological samples that can be preserved from wild animal species, testicular samples from healthy individuals offer additional possibilities to produce offspring, even after death. Testicular fragments can be cryopreserved using slow freezing or vitrification, but protocols vary according to the age or reproductive status of the donor. The present study demonstrated that solid surface vitrification was better than slow freezing for the preservation of testicular fragments from prepubertal collared peccaries. Specifically, a 6 M combination of dimethyl sulfoxide and ethylene glycol in the vitrification solution was the most efficient.

The cryopreservation of male gonadal tissue is critical to conserve genetic material and use it later via assisted reproduction. This study aimed to evaluate cryopreservation methods (slow freezing, SF; solid surface vitrification, SSV) as well as the optimal concentrations of intracellular cryoprotectants during the SSV of testicular tissue from prepubertal collared peccaries. Five pairs of testes were dissected on different days into small fragments (3 mm3) and allocated to a non-cryopreserved, a control group or one of three treatment groups: SF; SSV 3 M (1.5 M dimethyl sulfoxide [DMSO] plus 1.5 M ethylene glycol [EG]); or SSV 6 M (3 M DMSO plus 3 M EG). After one week of storage in liquid nitrogen, tissue samples were warmed and evaluated in terms of histology, viability, proliferative capacity potential, and DNA integrity. The scores for histological integrity and cellular damage for SF (2.08 ± 0.05 and 2.33 ± 0.07, respectively) were similar to the results found in SSV 6 M (1.93 ± 0.04 and 2.30 ± 0.07; p > 0.05). However, these scores were better when compared to SSV 3 M (1.87 ± 0.05 and 2.08 ± 0.06; p < 0.05). The percentage of cellular viability was around 57% after all preservation treatments (p > 0.05), which was lower than in the control group (88.8 ± 1.9%; p < 0.05). The SSV 6 M treatment was better than the other treatments regarding the proliferative capacity potential of spermatogonia cells (3.52 ± 0.03) (p < 0.05), although it was lower than in the control group (4.00 ± 0.12) (p < 0.05). Additionally, SSV 6 M led to the same DNA integrity (97.0 ± 0.7%) as in the control group (99.4 ± 0.3%). These collective findings suggest that the combination of SSV with 6 M cryoprotectants is the most efficient for the cryopreservation of testes from prepubertal collared peccaries.